Endothelial cell phenotypes display remarkable heterogeneity in health and disease. An important goal in vascular biology is to understand the molecular mechanisms underlying the spatial and temporal modulation of endothelial cell phenotypes. A more complete understanding of these mechanisms should provide a framework for tailoring and fine tuning therapeutic modalities in vascular disease states. One approach to this problem is to focus on a discrete signal transduction network as a tool for characterizing the dynamic nature of endothelial cell (dys)function. Thrombin signaling represents one such network. We have recently shown that thrombin is coupled to the activation of a number of different transcriptional networks and downstream target genes in endothelial cells. The overall goal of this project is to delineate the mechanisms by which thrombin signaling is regulated in space and time. In Specific Aim #1, the molecular basis of PAR-1-mediated induction of VCAM-1 in endothelial cells will be studied in more detail. To that end, the interactions between PKC-delta and NF-kB p65 and between PKC-zeta and GATA-2 will be characterized. Moreover, the role of p38 MAPK in the PAR-1-VCAM-1 signaling pathway will be determined. In Specific Aim #2, the role of PAR-1-coupled signaling pathways in mediating distinct gene programs in endothelial cells will be dissected. To that end, HUVEC will be pretreated with chemical inhibitors (of PI3K), infected with dominant negative (DN) adenoviruses (DN PKC-delta, PKC-zeta, NF-kB) or transfected with antisense/RNAi (against GATA-2, Egr-1). The cells will then be incubated in the presence of PAR-1 agonists and assayed for gene expression and cellular function. In Specific Aim #3, HUVEC will either be grown in co-culture or primed with physiological or pathophysiological stimuli. The preconditioned endothelial cells will then be incubated in the presence of PAR-1 agonists and assayed for gene expression and cellular function. In Specific Aim #4, the effect of PAR-1 agonists on the intact vasculature will be studied in Langendorff heart preparations. The results of these studies should yield valuable information about the spatial and temporal dynamics of endothelial cell activation, and in particular the role of PAR-1 signaling in modulating cellular phenotypes.